The von Hippel-Lindau (VHL) syndrome is a hereditary syndrome that predisposes affected patients to develop a variety of neoplasms including sporadic renal cell carcinomas, pheochromocytomas, and CNS hemangioblastomas. VHL-associated neoplasms are typically hypervascular and overproduce angiogenic peptides, such as vascular endothelial growth factor (VEGF), probably because the VHL gene product (pVHL) is a negative regulator of hypoxia-inducible mRNAs. pVHL is also known to form a complex (hCul-2-VBC) with human cullin-2 (hCul-2) through elongins B/C and possesses a tumor suppressor function in vivo. Interestingly, a frequently mutated region of pVHL in patients with VHL syndrome contains a binding site for elongins B/C and the mutations interfere with the formation of the hCul-2-VBC complex, suggesting that the inability to form the hCul-2-VBC complex plays a critical role in the pathogenesis of VHL syndrome. Recently, we found that hCul-2 is covalently modified by a single molecule of NEDD8, a novel ubiquitin-like protein which does not target proteins for proteolytic degradation by proteasome. This post-translational modification of hCul-2 by NEDD8 may regulate the formation or the function of the hCul-2- VBC complex. This proposal is designed to study the mechanism and biological function of NEDD8-modification, using hCul-2 as a model substrate. In particular, we will focus on the role of NEDD8- conjugation in the pathogenesis of VHL syndrome. The aims are to define: 1) the target Lys residue of hCul-2 which is covalently modified by NEDD8, 2) the relationship between phosphorylation and NEDD8-conjugation of hCul-2, 3) the effect of NEDD8- conjugation to hCul-2 on cell-cycle progression, 4) the role of NEDD8-conjugation to hCul-2 in the formation and subcellular localization of hCul-2-VBC complex and in the regulation of hypoxia-inducible mRNA, such as VEGF.